Millicell-CM® insert

Mcllwain tissue chopper (Brinkmann Instruments). Millicell-CM inserts (Millipore Billerica, MA). 

Plate two to three slices onto a Millicell-CM insert, and place each insert inside a 3 5-mm petri dish containing 1 mL of culture medium. Incubate at 34 °C in 5 % CO2 for up to 8 DIV. Medium has to be changed twice a week. Allow slices to equilibrate to the in vitro conditions for at least 4-6 DIV before treatments. 

Insert cartridges in the barrel of the Helios Gene Gun . Connect the Helios Gene Gun with the helium cylinder, and select an operating pressure of 160 psi. Place the barrel liner over the target (i.e., the dish containing the coverslip with attached cultured neurons or the petri dish containing the slice on the Millicell-CM insert) at a distance of 1.6 mm with a spacer. 

Remove the culture medium from a petri dish containing the coverslip with attached cells or the slices on the Millicell-CM insert and add 1 mL of 4 % PFA in PB. Fix for 1 h and wash 3x5 min in PBS. From this stage on cultures can be processed in multi-well plates by transferring them from a well to another with forceps. Isolated cells attached to coverslips can be directly handled as indicated, whereas organotypic cultures need to be separated from the Millicell-CM insert by carefully cutting the membrane around tissue slices and leaving enough membrane to allow manipulation. 

Then transfer a culture on a Millicell-CM insert in the microscope stage incubator, close the chamber lid and turn on the CO2 supply. Allow culture to equilibrate and then choose a microscope field with several transfected cells using the conventional fluorescence microscopy mode of the confocal microscope. 

Fig. 3.2(a). Vessels in which cells may be cultured. At the top left are three sizes of flask (125, 75 and 25 cm2) and at the bottom left three sizes of dish (9.5 and 3 cm diameter). To the right are shown 6, 12 and 24-well tissue culture trays and a 96-well microtitre plate. At the bottom right is an 8-chamber culture slide, (b) Millicell inserts for 6-well TC trays provide a semi-permeable growth surface. 

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